Pharmaceutical formulation comprising P1-(2&#39;-deoxycytidine 5&#39;-)P4-(uridine 5&#39;-) tetraphosphate

ABSTRACT

The present invention provides a method of treating edematous retinal disorders. The method comprises administration of a pharmaceutical formulation comprising a hydrolysis-resistant P2Y receptor agonist to stimulate the removal of pathological extraneous fluid from the subretinal and retinal spaces and thereby reduce the accumulation of said fluid associated with retinal detachment and retinal edema. The P2Y receptor agonist can be administered with therapeutic and adjuvant agents commonly used to treat edematous retinal disorders. The pharmaceutical formulation useful in this invention comprises a P2Y receptor agonist with enhanced resistance to extracellular hydrolysis, such as dinucleoside polyphosphate compounds, or hydrolysis-resistant mononucleoside triphosphates.

This application is a continuation-in-part of U.S. application Ser. No.09/774,752, filed Jan. 30, 2001 which issued as U.S. Pat. No. 6,596,725on Jul. 22, 2003; which is a continuation-in-part of U.S. applicationSer. No. 09/101,395, filed Jul. 10, 1998, which issued as U.S. Pat. No.6,348,589 on Feb. 19, 2002; which was the National Stage ofInternational Application No. PCT/US98/02702, filed Feb. 6, 1998,published Aug. 13, 1998 under PCT Article 21(2) in English; and was acontinuation-in-part of U.S. application Ser. No. 08/798,508, filed Feb.10, 1997, which issued as U.S. Pat. No. 5,837,861 on Nov. 17, 1998. AllU.S. applications and patents cited herein are specifically incorporatedherein by reference in their entirety.

TECHNICAL FIELD

This invention relates to a method of treating edematous retinaldisorders. Specifically, this invention relates to a method of removingpathological fluid accumulation in subretinal and intra-retinal spaces.

BACKGROUND OF THE INVENTION

The retinal pigment epithelium (RPE) lies in the back of the vertebrateeye and forms a barrier that separates the retina from the choroidalblood supply. A critical function of the RPE is to maintain and regulatethe hydration of the subretinal space, the extracellular volume thatexists between the retina and the RPE. (Marmor, pp. 3-12, in TheRetinal, Pigment Epithelium, Eds. M. F. Marmor and T. J. Wolfensberger,Oxford University Press, New York, (1998)). This function is achieved bythe regulated transport of fluid, ions, and metabolites between thesubretinal space and the choroidal blood supply. (Marmor, pp. 420-438,in The Retinal Pigment Epithelium, Eds. M. F. Marmor and T. J.Wolfensberger, Oxford University Press, New York, (1998); Pederson, pp.1955-1968, in Retina, Ed. S. J. Ryan, Mosby, St. Louis, (1994)). Likeall epithelia, the RPE contains two functionally and anatomicallydistinct membranes: an apical membrane that faces the retina, and abasolateral membrane that faces the choroidal blood supply. In thenormal retina, fluid is absorbed across the RPE in the direction of thesubretinal space to the choroid. This active absorption of fluid by theRPE, often referred to as the “RPE pump,” plays a critical role inmaintaining proper attachment of photoreceptors to the apical membraneof the RPE by pumping fluid out of the retinal spaces. (Marmor, pp.1931-1954, in Retina, Ed. S. J. Ryan, Mosby, St. Louis, (1994); Hughes,et al., pp. xvii, 745, in The Retinal Pigment Epithelium, Eds. M. F.Marmor and T. J. Wolfensberger, Oxford University Press, New York,(1998)).

Retinal detachment is characterized by abnormal accumulation of fluid inthe subretinal space leading to detachment of the retina from theunderlying retinal pigment epithelium. Retinal edema refers to abnormalaccumulation of fluid within the retina itself. Retinal detachment oredema in the central part of the retina (macula) produces significantloss of vision, and can ultimately lead to irreversible blindness.(Yanoff and Duker, Ophthalmology, Mosby, Philadelphia, (1999);Wilkinson, et al., Michels' Retinal Detachment, 2^(nd) ed. Mosby, St.Louis, (1997)) A wide variety of ocular pathologies can result inretinal detachment or retinal edema. The most common type of retinaldetachment is rhegmatogenous retinal detachment, which occurs as aresult of single or multiple tears or holes in the retina that permitliquefied vitreous to enter into the subretinal space and create aretinal detachment.

There are no pharmacological approaches employed in the treatment ofrhegmatogenous retinal detachment (RRD). The only current treatments forRRD are surgical (scleral buckling, pneumatic retinopexy, orvitrectomy). (Wilkinson, Michels' Retinal Detachment, 2nd ed., Mosby,St. Louis, (1997)). There are two vital components for successful RRDsurgery: reattachment of the retina and repair of the retinal break. Theprincipal difference among the three surgical techniques for treatingRRD is in the method employed to facilitate retinal reattachment.

Scleral buckle uses an extraocular buckle (usually a silicone sponge orsolid silicone) that is sewn to the sclera towards the detached retina(Wilkinson, et al., Michels' Retinal Detachment, 2nd ed., Mosby, St.Louis (1997)). The retina usually reattaches over a period of a fewdays, but may take up to a few weeks. The surgeon may elect to drain thesubretinal fluid at the time of operation by inserting a needle throughthe sclera, choroid, and RPE. In general, the buckle remains permanentlysewn to the sclera. In pneumatic retinopexy, a gas bubble is injecteddirectly into the vitreous, and the head is positioned so that the gasbubble acts as a tamponade and covers the retinal break. (Tomambe andHilton, Ophthalmology 96(6):772-83 (1989)). The subretinal fluid usuallyresolves within 1-2 days, but precise head positioning is required toinsure that the bubble covers the retinal break. (Tomambe, 10 et al.,Am. J. Ophthalmol. 127(6):741-3 (1999)). Vitrectomy is usually used forcomplex RRD associated with vitreous traction or hemorrhage, but isoccasionally used for simple RRD (Chang, pp. 8.34.1-8.34.6, inOphthalmology, Eds. M. Yanoff and J. S. Duker, Mosby, Philadelphia,(1999)). The procedure involves making three small incisions through thesclera to allow the introduction of instruments in the vitreous cavity.The vitreous is removed and replaced with a special saline solution.Depending on the type and cause of the detachment, a variety ofinstruments and techniques are then used to reattach the retina. Forsimple detachments, the retina is flattened via anterior drainage of thesubretinal space by insertion of a needle through the retinal tear.

Scleral buckle and vitrectomy often require general anesthesia and caninvolve hospitalization. Pneumatic retinopexy is usually done in thephysician's office, but requires patient compliance for success. (Hiltonand Tornambe, Retina 11(3):285-94 (1991); Hilton and Brinton, pp.2093-2112, in Retina, Ed. Stephen J. Ryan, Mosby, Philadelphia, (1999);Han, et al., Am. J. Ophthalmol. 126(5):658-68 (1998)). Depending on thesurgical technique and the surgeon, success rates can vary following asingle surgery, with lower rates for pneumatic retinopexy and higherrates for scleral buckle. (Tomambe, et al., Am. J. Ophthalmol.127(6):741-3 (1999); Han, et al., Am. J Ophthalmol. 126(5):658-68(1998)). The success of retinal detachment surgery is measured in termsof retinal reattachment at any point following surgery (ranging fromhours to weeks). Parameters such as visual outcome and patientquality-of-life are not used to assess success of retinal detachmentsurgery.

The conditions that are commonly associated with the more severe formsof intra-retinal edema are diabetic macular edema, exudative age-relatedmacular degeneration (AMD) and clinically significant cystoid macularedema. (Jampol and Po, pp. 999-1008, in Retina, Ed. S. J. Ryan, Mosby,St. Louis, (1994)). Other pathological conditions associated withabnormal fluid accumulation in intra-retinal or subretinal spacesinclude uveitis, central and branch vein occlusion, retinitispigmentosa, central serous retinopathy, CMV retinitis, and choroidalmelanoma. Physical trauma associated with ocular injury followingcertain surgical procedures (such as cataract surgery) can also produceretinal detachment or edema. (Ahmed and Ai, pp. 8.34.1-8.34.6, inOphthalmology, Eds. M. Yanoff and J. Duker, Mosby, Philadelphia,(1999)).

Intra-retina accumulation of fluid in the macula results in decreasedvisual acuity and is the most common cause of vision loss in patientswith diabetic retinopathy, AMD and other ischemic retinopathies such asbranch and central retinal vein occlusion. (Jampol and Po, pp. 999-1008,in Retina, Ed. Stephen J. Ryan, Mosby, St. Louis, (1994); Kent, et al.,Br. J Ophthalmol. 84(5):542-5 (2000)). Macular edema is a frequentcomplication of uveitis and is commonly seen in patients with retinitispigmentosa. (Rothova, et al., Br. J Ophthalmol. 80(4):332-6 (1996);Fetkenhour, et al., Trans. Am. Acad. Ophthalmol. Otolaryngol. 83(3)Pt 1:OP515-21 (1977)). Macular edema is also a major cause of decreasedvision following intraocular surgery (called cystoid macular edema).(Miyake, Surv. Ophthalmol. 28 Suppl:554-68 (1984)). Accumulation ofintra-retina fluid is believed to result from a breakdown of the innerand/or outer blood-retinal barrier. (Kent, et al., Br. J. Ophthalmol.84(5): 542-5 (2000)). The inner retinal barrier consists of endothelialcells of the retinal vasculature and the outer barrier comprises theretinal pigment epithelium. Breakdown of the blood-retinal barrier canresult in focal leakage of fluid from the vasculature and fluidaccumulation within retinal layers or in the subretinal space. Thepresent treatments for retinal edema include systemic and topicaladministrations of corticosteroid, acetazolamide, and non-steroidalanti-inflammatory drugs, as well as surgical options such as vitrectomy,grid, and focal laser photocoagulation. These therapies show limitedutility in patients.

Although modem day RRD surgery has a relatively high success rate(60-90%), it is thought that a pharmaceutical composition that canreattach the retina in cases where surgery failed would be of enormouspatient benefit. In addition, if the pharmaceutical composition canreattach the retina in the absence of surgical intervention, it would bemost therapeutically useful, particularly in the treatment ofrhegmatogenous retinal detachment.

A number of pharmacological and surgical approaches are employed totreat cystoid and diabetic macular edema, but they are generallyconsidered empirical and often ineffective. Non-specificanti-inflammatory treatment is used for all types of macular edema,except in cases associated with ischemic retinopathies in which lasertreatment is indicated (Kent, et al., Br. J. Ophthalmol. 84 (5):542-5(2000)). Corticosteroids are frequently used to treat macular edema, butwere shown to be ineffective in a randomized, placebo controlled study(Flach, et al., Am. J. Ophthalmol. 103(4):479-86 (1987); Flach, et al.,Ophthalmology 97(10):1253-8 (1990)). Acetazolamide also alleviatescertain types of macular edema and is postulated to work via activationof the RPE pump, but systemic tolerance to acetazolamide is poor. (Cox,et al., Arch. OphthaZmoZ. 106(g):1190-5 (1988)). Focal or grid laserphotocoagulation is commonly used to reduce retinal vascular leakageassociated with diabetic retinopathy, and is useful in limited cases.(Ip, et al., In Ophthalmology, London; Philadelphia: Mosby, 8.4.1-8.4.2(1999); The Diabetic Retinopathy Study Research Group, Ophthalmology 88(7):583-600 (1981); Early Treatment Diabetic Retinopathy Study ResearchGroup, Arch. Ophthalmol. 103(12):1796-806 (1985); The Branch VeinOcclusion Study Group, Am. J. Ophthalmol. 98(3):271-82 (1984)). Inaddition, vitrectomy is employed to treat diabetic retinopathiesassociated with vitreal hemorrhages and/or vitreoretinal traction.(Wilkinson, et al, Michels' Retinal Detachment, 2nd ed., Mosby, St.Louis, (1997)). There remains a large unmet medical need for a safe,effective treatment of macular edema. (Kent, et al., Br. J. Ophthalmol.84(5):542-5 (2000)).

Peterson, et al. (J. Neurosci. 17:2324-37 (1997)) suggest that UTP (orperhaps ATP) could be used therapeutically to reduce the pathologicalaccumulation of fluid in the subretinal space. However, both ATP and UTPare rapidly degradeable by ubiquitous extracellular nucleotidases.Therefore, in order for ATP and UTP to be efficacious in the treatmentof retinal detachment, these compounds need to be delivered directlyinto the subretinal space. Drug delivery into the subretinal space iswidely regarded to be unacceptably risky for patients because itinvolves the insertion of a needle between the retina and RPE, which canresult in complications and blindness. In order for ATP or UTP to betherapeutically useful, it must be delivered into the intravitrealcavity, which is a much less invasive procedure. However, in order forATP or UTP to reach the RPE apical membrane, it must diffuse across theretina. It is unknown if intravitreal ATP or UTP is degraded by the timeit reaches the RPE apical membrane and therefore effective instimulating retinal reattachment. The present examples show thatintravitreal UTP is ineffective in stimulating retinal reattachment andthat the present method is effective in stimulating retinalreattachment.

SUMMARY OF THE INVENTION

Pharmaceutical compositions and methods of use thereof for stimulatingremoval of extraneous fluid in the retina or subretinal space in asubject in need of such treatment are disclosed. The methods andcompositions disclosed in the present invention are used to stimulateremoval of extraneous intra-retinal or subretinal fluid for any reason,including, but not limited to, primary and adjunctive treatments ofrhegmatogenous retinal detachment, serous retinal detachment, all formsof cystoid macular edema (uveitis, post-surgical, central and branchvein occlusion, and inherited retinal diseases such as retinitispigmentosa), and all forms of retinal and macular edema (proliferativeand non-proliferative, exudative age-related macular degeneration, andretinopathy of prematurity.)

The present invention discloses methods of treating a subject withedematous retinal disorders such as retinal detachment or retinal edemaby administering a pharmaceutical composition comprising ahydrolysis-resistant P2Y receptor agonist via systemic or topicaladministration such as intravitreal injection, intravitreal sustainedrelease or delivery, ocular surface instillation, transcleral injectionor infusion, or systemic injection or infusion.

The pharmaceutical formulations useful in this invention compriseadenine-, uridine-, and cytidine-containing dinucleoside polyphosphates,and derivatives thereof, and hydrolysis-resistant monucleosidetriphosphates, which are selective agonists of the P2Y receptor onepithelial cells of the retinal pigment epithelium.

DESCRIPTION OF THE FIGURES

FIG. 1 represents cellular localization of P2Y₂ receptor mRNA in freshfrozen cross sections of albino rabbit retina/RPE/choroid tissue usingnonisotopic in situ hybridization techniques. Specifically, arepresentative in situ hybridization result from antisense and sensedigoxigenin (DIG)-labeled riboprobes engineered based on the P2Y₂receptor mRNA sequence is shown. GCL: ganglion cell layer. IPL: innerplexiform layer. INL: inner nuclear layer. ONL: outer nuclear layer. IS:inner segments. OS: outer segments.

FIG. 2 represents the effects of UP₄dC (INS37217) versus UTP oncytosolic calcium mobilization in 1321N1 cells overexpressing P2Y₂receptor.

FIG. 3 represents the effect of UP₄dC versus UTP on inositol phosphategeneration in 1321N1 cells overexpressing P2Y₂ receptor.

FIG. 4 represents the effects of UP₄dC on fluid absorption in humanfetal RPE.

FIG. 5 represents the effects of UP₄dC on the magnitude and direction offluid transport in bovine RPE.

FIG. 6 represents the metabolism rates of UP₄dC and UTP from freshlyisolated pig retinal tissue.

FIG. 7 shows the effects of subretinal UP₄dC on the reabsorption ofsubretinal blebs. Subretinal blebs were created by injecting MPBSsolution into the subretinal space with or without UP₄dC (1 mM).Summarized results (mean ±SEM) show that UP₄dC increased the rate ofclearance of subretinal blebs when compared with vehicle control.

FIGS. 8 (A-C) shows the effects of intravitreal UP₄dC on thereabsorption of subretinal blebs. MPBS solution was injected into thesubretinal space to create subretinal blebs, followed immediately by anintravitreal injection of MPBS solution with or without UP₄dC (12 mM,1.4 mM, and 0.15 mM). Summarized results show that UP₄dC administered at12 and 1.4 mM, but not 0.15 mM, increased the rate of clearance ofsubretinal blebs when compared with vehicle control.

FIG. 9 shows a significant difference (p<0.05) between UP₄dC (open bars)and vehicle control (placebo, solid bars) on the scoring of subretinalblebs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of increasing fluid absorptionacross the retinal pigment epithelium (RPE) to facilitate the removal ofextraneous intraretinal or subretinal fluid from the posterior portionof the eye to treat diseases that lead to retinal detachment and retinaledema. The apical (retinal-facing) membrane of the RPE contains P2Yreceptors that can be activated to stimulate fluid transport across theRPE in the direction from the subretinal space to the choroidal bloodsupply, so as to facilitate the removal of subretinal fluid in retinaldetachment. The present method comprises administering to a subject aP2Y receptor agonist that is hydrolysis-resistant in an amount effectiveto stimulate the removal of pathological fluid accumulation inintra-retinal and subretinal spaces associated with edematous retinaldisorders. Activation of P2Y receptors by such agonists is associatedwith elevated intracellular calcium levels and increased fluid transportacross the RPE.

The P2Y receptor agonist is administered with or without othertherapeutic and adjuvant agents commonly used to treat or manage retinaldetachment and retinal edema. An effective dose is the amount of suchagonist necessary to activate P2Y receptors at the retinal-facing(apical) membrane of retinal pigment epithelial cells and to enhancefluid absorption (retinal-to-choroidal direction) across the RPE.

The method of the present invention is useful for the prevention,management and/or treatment of all disorders associated with retinaldetachment and retinal edema, including but not limited torhegmatogenous retinal detachment, serous retinal detachment, all formsof cystoid macular edema (uveitis, post-surgical, central and branchvein occlusion, and inherited retinal diseases such as retinitispigmentosa), and all forms of retinal and macular edema (proliferativeand non-proliferative diabetic macular edema, exudative age-relatedmacular degeneration, and retinopathy of prematurity).

This invention provides a method of administering to a subject apharmaceutical composition comprising a P2Y receptor agonist forremoving pathological fluid accumulation in subretinal and intra-retinalspaces. P2Y receptor agonists useful for the present method arehydrolysis-resistant compounds such that they are not degraded by thetime they reach the RPE apical membrane and therefore they are effectivein stimulating retinal reattachment. P2Y receptor agonists useful forthe present method include dinucleoside polyphosphates,hydrolysis-resistant mononucleoside triphosphates and their analogues,which activate P2Y₁, P2Y₂, P2Y₄, P2Y₆, and/or P2Y₁₁, and preferably P2Y₂receptors.

Description of Compounds

Dinucleoside polyphosphates useful for this invention are compounds ofgeneral Formula I or the pharmaceutically acceptable non-toxic saltsthereof:

wherein:

X is oxygen, methylene, halomethylene, dihalomethylene (with halo beingfluoro or chloro preferred), or imido;

n=0, 1 or 2;

m=0, 1 or 2;

n+m=0, 1,2,3, or 4;

Z=OH or H;

Z′=OH or H;

Y=OH, N₃ or H;

Y′=OH, N₃ or H; and

B and B′ are each independently a purine residue or a pyrimidineresidue, as defined in Formula Ia or Ib, linked through the 9- or1-position, respectively;

wherein:

R₁ is selected from the group consisting of: hydrogen, fluoro, chloro,bromo, cyano, azido, amino, N-alkylamino, N,N-dialkylamino,N-cycloalkylamino, N-aralkylamino, N-arylamino, N-acylamino (amide),N-alkyl-N-acylamino (N-alkyl amide), N-cycloalkyl-N-acylamino(N-cycloalkyl amide), N-aralkyl-N-acylamino (N-aralkyl amide),N-aryl-N-acylamino (N-aryl amide), alkyloxy, aralkyloxy, aryloxy,alkylthio, arylthio, and aralkylthio, wherein such a substituent on thenitrogen, oxygen, or sulfur contains up to a maximum of 20 carbon atoms,with or without unsaturation; optionally, up to two heteroatoms selectedfrom the group consisting of oxygen, NH, and sulfur are substituted inplace of carbon units in such a substitution; optionally, saidN,N-dialkylamino groups are linked to form a heterocycle of 3 to 7members;

R₂ is hydroxy, alkenyl, oxo, amino, mercapto, thione, alkylthio,arylthio, aralkylthio, acylthio, alkyloxy, aryloxy, aralkyloxy, acyloxy,N-alkylamino, N,N-dialkylamino, N-cycloalkylamino, N-aralkylamino,N-arylamino, N-acylamino (amide), N-alkyl-N-acylamino (N-alkyl amide),N-cycloalkyl-N-acylamino (N-cycloalkyl amide), N-aralkyl-N-acylamino(N-aralkyl amide), N-aryl-N-acylamino (N-aryl amide), or a heterocyclicmoiety containing 3 to 10 carbons atoms;

R_(x) is O, H or is absent;

R₂ and R_(x) are optionally taken together to form a 5-membered fusedimidazole ring of a 1,N⁶-etheno adenine derivative, optionallysubstituted on one or both of the 4- or 5-positions of the etheno moietywith alkyl, aryl or aralkyl moieties as defined below;

R₃ is selected from the group consisting of hydrogen, fluoro, chloro,bromo, cyano, azido, amino, N-alkylamino, N,N-dialkylamino,N-cycloalkylamino, N-aralkylamino, N-arylamino, N-acylamino (amide),N-alkyl-N-acylamino (N-alkyl amide), N-cycloalkyl-N-acylamino(N-cycloalkyl amide), N-aralkyl-N-acylamino (N-aralkyl amide),N-aryl-N-acylamino (N-aryl amide), alkyloxy, aralkyloxy, aryloxy,alkylthio, arylthio, and aralkylthio, wherein such a substituent on thenitrogen, oxygen, or sulfur contains up to a maximum of 20 carbon atoms,with or without unsaturation; optionally, up to two heteroatoms selectedfrom the group consisting of oxygen, NH, and sulfur are substituted inplace of carbon units in such a substitution; optionally, saidN,N-dialkylamino groups are linked to form a heterocycle of 3 to 7members; or absent;

J is carbon or nitrogen, with the provision that when J is nitrogen, R₃is not present;

wherein:

R₄ is hydroxy, oxo, mercapto, thione, amino, cyano, arylalkoxy,alkylthio, alkoxy, N-alkylamino, N,N-dialkylamino, N-cycloalkylamino,N-aralkylamino, N-arylamino, N-acylamino (amide), N-alkyl-N-acylamino(N-alkyl amide), N-cycloalkyl-N-acylamino (N-cycloalkyl amide),N-aralkyl-N-acylamino (N-aralkyl amide), N-aryl-N-acylamino (N-arylamide), or a heterocyclic moiety containing 3 to 10 carbons atoms;optionally, said N,N-dialkylamino groups are linked to form aheterocycle of 3 to 7 members;

R₅ is hydrogen, acetyl, benzoyl, alkyl, alkanoyl, aroyl, or absent;

R₆ is hydroxy, oxo, mercapto, thione, amino, cyano, arylalkoxy,alkylthio, alkoxy, aryloxy, N-alkylamino, N,N-dialkylamino,N-cycloalkylamino, N-aralkylamino, N-arylamino, N-acylamino (amide),N-alkyl-N-acylamino (N-alkyl amide), N-cycloalkyl-N-acylamino(N-cycloalkyl amide), N-aralkyl-N-acylamino (N-aralkyl amide),N-aryl-N-acylamino (N-aryl amide), or a heterocyclic moiety containing 3to 10 carbons atoms; optionally said N,N-dialkylamino groups are linkedto form a heterocycle; wherein said N-, O- or S-alkyl group isoptionally linked to N³ to form a saturated or unsaturated heterocyclicring of 5 to 7 members, with or without substituents; or

R₅ and R₆ are taken together to form a 5-membered fused imidazole ringbetween positions 3 and 4 of the pyrimidine ring and form a 3,N⁴-ethenocytosine derivative, wherein said etheno moiety is optionallysubstituted on one or both of the 4- or 5-positions with a moietyselected from the group consisting of: alkyl, aryl, aralkyl, aryloxy,alkyloxy, and aralkoxy;

R₇ is selected from the group consisting of: hydrogen, hydroxy, cyano,nitro, alkyl, aralkyl, alkenyl, aralkenyl, alkynyl, aryl, aralkynyl,halogen, CF₃, allylamino, bromoyinyl, ethyl propenoate, propenoic acidand alkyl or aryl esters thereof; or

R₆ and R₇ optionally form a 5 or 6-membered saturated or unsaturatedring bonded through N or O or S at R₆, such ring optionally containsalkyl, aralkyl or aryl substituents;

R₈ is selected from the group consisting of hydrogen, fluoro, chloro,bromo, cyano, azido, amino, carboxy, carbalkoxy, carbobenzyloxy,carboxamido, N-alkylcarboxamido, N-alkylamino, N,N-dialkylamino,N-cycloalkylamino, N-aralkylamino, N-arylamino, N-acylamino (amide),N-alkyl-N-acylamino (N-alkyl amide), N-cycloalkyl-N-acylamino(N-cycloalkyl amide), N-aralkyl-N-acylamino (N-aralkyl amide),N-aryl-N-acylamino (N-aryl amide), alkyloxy, aralkyloxy, aryloxy,alkylthio, arylthio, or aralkylthio, wherein such a substituent on thenitrogen, oxygen, or sulfur contains up to a maximum of 20 carbon atoms,with or without unsaturation; optionally, up to two heteroatoms selectedfrom the group consisting of oxygen, NH, and sulfur are substituted inplace of carbon units in such a substitution; optionally, saidN,N-dialkylamino groups are linked to form a heterocycle of 3 to 7members;

wherein the alkyls, alkenyls, and alkynyls are straight-chain, branchedor cyclic; substituted or unsubstituted.

Examples of the substituted adenine derivatives of Formula Ia includeadenine 1-oxide; 1,N⁶-(4- or 5-substituted etheno) adenine;N⁶-substituted adenine; or N-substituted 8-aminoadenine, where R′ of the6- or 8-HNR′ groups are chosen from among: arylalkyl groups with thearyl moiety optionally functionalized as described below, alkyl, andalkyl groups with functional groups therein, such as:([6-aminohexyl]carbamoylmethyl)-, and ω-acylated-amino (hydroxy, thiolor carboxy)alkyl derivatives where the acyl group is chosen from among,but not limited to, acetyl, trifluoroacetyl, benzoyl,substituted-benzoyl, etc., or the carboxylic moiety is present as itsester or amide derivative, for example, the ethyl or methyl ester or itsmethyl, ethyl or benzamido derivative. The ω-amino(hydroxy, thiol)moiety can be alkylated with a C₁₋₄ alkyl group.

Formula I is drawn without stereochemical properties shown, however, thefuranosyl moieties are preferably in the D-configuration, but can be L-,or D- and L-. Preferably, the nucleoside residues include the sugarmoieties selected from the group consisting of: ribofuranosyl,arabinofuranosyl, 2′-deoxyribofuranosyl, 3′-deoxyribofuranosyl,2′,3′-dideoxyribofuranosyl, xylofuranosyl, 2′-deoxyxylofuranosyl andlyxofuranosyl; and these sugar moieties can be in the alpha- or beta-and D- or L-configurations, but most preferably are in thebeta-D-configuration.

In the general structures of Formulae I, Ia and Ib above, the dottedlines in the 2- to 6-positions are intended to indicate the presence ofsingle or double bonds in these positions; the relative positions of thedouble or single bonds being determined by whether the R₄, R₅ and R₆substituents are capable of keto-enol tautomerism.

In the general structures of Formula I above, the acyl groups comprisealkanoyl or aroyl groups. Preferably, the alkyl groups contain 1 to 8carbon atoms, particularly 1 to 4 carbon atoms optionally substituted byone or more appropriate substituents, as described below. The arylgroups including the aryl moieties of such groups as aryloxy arepreferably phenyl groups optionally substituted by one or moreappropriate substituents, as described below. The above-mentionedalkenyl and alkynyl groups preferably contain 2 to 8 carbon atoms,particularly 2 to 6 carbon atoms, e.g., ethenyl or ethynyl, optionallysubstituted by one or more appropriate substituents as described below.

Appropriate substituents on the above-mentioned alkyl, alkenyl, alkynyl,aralkyl, and aryl groups are selected from halogen, hydroxy, C₁₋₄alkoxy, C₁₋₄ alkyl, C₆₋₁₂ aryl, C₆₋₁₂ arylalkoxy, carboxy, cyano, nitro,sulfonamido, sulfonate, phosphate, sulfonic acid, amino and substitutedamino wherein the amino is singly or doubly substituted by a C₁₋₄ alkyl,and when doubly substituted, the alkyl groups optionally being linked toform a heterocycle.

Dinucleoside polyphosphates of general Formula I include dinucleosidetetraphosphates selected from the group consisting of: P¹,P⁴-di (uridine5′-)tetraphosphate (UP₄U); P¹-(cytidine 5′-)P⁴-(uridine5′-)tetraphosphate (CP₄U); P¹,P⁴-di(adenosine 5′-) tetraphosphate;P¹-(adenosine 5′-)P⁴-(uridine 5′-)tetraphosphate; P¹-(adenosine 5′-)P⁴-(cytosine 5′-) tetraphosphate;P¹,P⁴-di(ethenoadenosine)tetraphosphate; P¹-(uridine 5′-)p⁴-(thymidine5′-)tetraphosphate; P¹-(adenosine 5′-)P⁴-(inosine 5′-)tetraphosphate;P¹,P⁴-di(uridine 5′-)P²,P³-methylenetetraphosphate; P¹,P⁴-di(uridine5′-)P²,P³-difluoromethyl -enetetraphosphate; P¹,P⁴-di(uridine5′-)P²,P³-imidotetraphosphate; P¹,P⁴-di(4-thiouridine5′-)tetraphosphate; P¹,P⁴-di(3,N⁴-ethenocytidine 5′-)tetraphosphate; P¹,P⁴-di(imidazo[1,2-c]pyrimidine-5(6H)-one-2-(3-nitro)-phenyl-6-β-D-ribofuranoside5′-)tetraphosphate; P¹-(inosine 5′-)P⁴-(uridine 5′-)tetraphosphate;P¹-(4-thiouridine 5′-) P⁴-(uridine 5′-)tetraphosphate; P¹-(cytosineβ-D-arabinofuranoside 5′-)P⁴-(uridine 5′-)tetraphosphate; P¹-(uridine5′-)P⁴-(xanthosine 5′-)tetraphosphate; P¹-(2′-deoxyuridine5′-)P⁴-(uridine 5′-)tetraphosphate; P¹-(3′-azido-3′-deoxythymidine5′-)P⁴-(uridine 5′-)tetraphosphate; P¹,P⁴-di(3′-azido-3′-deoxythymidine5′-)tetraphosphate; 2′(3′)-benzoyl-P¹,P⁴-di(uridine 5′-)tetraphosphate;P¹,P⁴-di(2′(3′)-benzoyl uridine 5′-)tetraphosphate;P¹-(2′-deoxyguanosine 5′-)P⁴-(uridine 5′-)tetraphosphate;P¹-(2′-deoxyadenosine 5′-)P⁴-(uridine 5′-)tetraphosphate;P¹-(2′-deoxyinosine 5′-)P⁴-(uridine 5′-)tetraphosphate;P¹-(2′-deoxycytidine 5′-)P⁴-(uridine 5′-)tetraphosphate (dCP₄U);P¹-(4-thiouridine 5′-)P⁴-(2′-deoxyuridine 5′-)tetraphosphate;P¹-(8-azaadenosine-5′-)P⁴-(uridine 5′-)tetraphosphate;P¹-(6-mercaptopurineriboside 5′-)P⁴-(uridine 5′-)tetraphosphate;P¹-(6-mercaptopurineriboside 5′-)P⁴-(2′-deoxyuridine 5′-)tetraphosphate;P¹-(4-thiouridine 5′-)P⁴-(arabinocytidine 5′-)tetraphosphate;P¹-(adenosine 5′-) P⁴-(4-thiomethyluridine 5′-)tetraphosphate;P¹-(2′-deoxyadenosine 5′)p⁴-(6-thiohexylpurineriboside5′-)tetraphosphate, and P¹-(6-octyloxypurineriboside 5′-)P⁴-(uridine5′-)tetraphosphate. UP₄U and dCP₄U are preferred compounds.

Dinucleoside polyphosphates of general Formula I also includedinucleoside triphosphates selected from the group consisting of:P¹,P³-di(uridine 5′-)triphosphate (UP₃U); P¹-(cytidine 5′-)P³-(uridine5′-)triphosphate; P¹,P³-di(adenosine 5′-)triphosphate; P¹-(adenosine5′-)P³-(uridine 5′-)triphosphate; P¹-(adenosine 5′-)P³-(cytidine5′-)triphosphate; P¹,P³-di(ethenoadenosine)triphosphate; P¹-(uridine5′-)P³-(thymidine 5′-)triphosphate; P¹-(adenosine 5′-)P³-(inosine5′-)triphosphate; P¹,P³-di(uridine 5′-)P²,P³-methylenetriphosphate;P¹,P³-di(uridine 5′-)P²,P³-difluoromethylenetriphosphate;P¹,P³-di(uridine 5′-)P²,P³-imidotriphosphate; P¹,P³-di(4-thiouridine5′-)triphosphate; P¹,P³-di(3,N⁴-ethenocytidine 5′-)triphosphate; P¹,P³-di(imidazo[1,2-c]pyrimidine-5(6H)-one-2-(3-nitro)-phenyl-6-β-D-ribofuranoside5′-)triphosphate; P¹-(inosine 5′-)P³-(uridine 5′-)triphosphate;P¹-(4-thiouridine 5′-)P³-(uridine 5′-)triphosphate; P¹-(cytosineβ-D-arabinofuranoside 5′-)P³-(uridine 5′-)triphosphate; P¹-(uridine5′-)P³-(xanthosine 5′-) triphosphate; P¹-(2′-deoxyuridine5′-)P³-(uridine 5′-)triphosphate; P¹-(3′-azido -3′-deoxythymidine 5′-)P³-(uridine 5′-)triphosphate; P¹,P³-di(3′-azido-3′-deoxythymidine 5′-)triphosphate; 2′(3′)-benzoyl-P¹,P³-di(uridine 5′-)triphosphate;P¹,P³-di(2′(3 ′)-benzoyl uridine 5′-)triphosphate; P¹(2′-deoxyguanosine5′-)P³-(uridine 5′-)triphosphate; P¹-(2′-deoxyadenosine 5′-)P³-(uridine5′-)triphosphate; P¹-(2′-deoxyinosine 5′-)P³-(uridine 5′-) triphosphate;P¹-(2′-deoxycytidine 5′-)P³-(uridine 5′-)triphosphate; P¹-(4-thiouridine5′-)P³-(2′-deoxyuridine 5′-)triphosphate;P¹-(8-azaadenosine-5′-)P³-(uridine 5′-)triphosphate;P¹-(6-mercaptopurine riboside 5′-)P³-(uridine 5′-)triphosphate;P¹-(6-mercaptopurineriboside 5′-)P³-(2′-deoxyuridine 5′-)triphosphate;P¹-(4-thiouridine 5′)P³-(arabinocytidine 5′-)triphosphate; P¹-(adenosine5′-)P³-(4-thiomethyluridine 5′-) triphosphate; P¹-(2′-deoxyadenosine5′-)P³-(6-thiohexylpurine riboside 5′-)triphosphate, andP¹-(6-octyloxypurineriboside 5′-)P³-(uridine 5′-)triphosphate.

Furthermore, dinucleoside polyphosphates of general Formula I includecompounds selected from the group consisting of: P¹-(uridine5′-)P²-(4-thiouridine 5′-)diphosphate; P¹,P⁵-di(uridine5′-)pentaphosphate; and P¹,P⁶-di(uridine 5′-)hcxaphosphate.

A hydrolysis-resistant P2Y agonist is a nucleotide with a modifiedphosphate ester backbone, e.g. a methylene, imido or other group thatprotects the phosphate ester bonds from being readily hydrolyzed.Dinucleotides are in general resistant to hydrolysis due to lack of aterminal phosphate group. Certain dinucleotides are especially resistantto hydrolysis. For example, P¹-(2′-deoxycytidine 5′)-P⁴-(uridine5′-)tetraphosphate is more resistant in comparison with P¹,P⁴-di(uridine5′-)tetraphosphate.

Furthermore, groups placed on or in the phosphate chain of amononucleotide impart some stability against hydrolysis, e.g. simplealkyl phosphate esters (methyl, ethyl, benzyl, etc.) or a thio group(e.g. UTPγS). Useful hydrolysis-resistant mononucleoside triphosphatesfor the present method include 5′-UTPγS, [79049-97-1], CA Index Name:Uridine 5′-trihydrogen diphosphate, P′-anhydride with phosphorothioicacid; 5′-CTPγS, [439919-14-9], CA Index Name: Cytidine 5′-trihydrogendiphosphate, P′-anhydride with phosphorothioic acid; 5′-TTPγS,[439919-15-0], CA Index Name: Thymidine 5′-trihydrogen diphosphate,P′-anhydride with phosphorothioic acid; 5′-GTPγS, [37589-80-3], CA IndexName: Guanosine 5′-trihydrogen diphosphate, P′-anhydride withphosphorothioic acid; 5′-CP₂NHP, [439919-16-1], CA Index Name:5′-Cytidylic acid, monoanhydride with imidodiphosphoric acid; 5′-TP₂NHP,[439919-17-2], CA Index Name: 5′-Thymidylic acid, monoanhydride withimidodiphosphoric acid; 5′-UP₂NHP, [82145-58-2], CA Index Name:5′-Uridylic acid, monoanhydride with imidodiphosphoric acid; 5′-GP₂NHP,[34273-04-6], CA Index Name: 5′-Guanylic acid, monoanhydride withimidodiphosphoric acid; 5′-AP₂NHP, [25612-73-1], CA Index Name:5′-Adenylic acid, monoanhydride with imidodiphosphoric acid; 5′-ATPγS,[35094-46-3], CA Index Name: Adenosine 5′-trihydrogen diphosphate,P′-anhydride with phosphorothioic acid; α,β-methylene 5′-ATP,[7292-42-4], CA Index Name: Adenosine, 5′-[hydrogen[[hydroxyphosphonooxyphosphinyl]methyl]phosphonate]; β,γ-methylene5′-ATP, [3469-78-1], CA Index Name: 5′-Adenylic acid, monoanhydride withmethylenebis[phosphonic acid]; 5′-ATPαS, [29220-54-0], CA Index Name:Adenosine, 5′→P″-ester with thiotriphosphoric acid;β,γ-difluoromethylene 5′-UTP, 5′-Uridylic acid, monoanhydride withdifluoromethylenebis[phosphonic acid]; β,γ-methylene 5′-UTP,[71850-06-1], CA Index Name: 5′-Uridylic acid, monoanhydride withmethylenebis[phosphonic acid]; and β,γ-dichloromethylene 5′-UTP;5′-Uridylic acid, monoanhydride with dichloromethylenebis[phosphonicacid].

The present invention also provides a novel composition chosen fromcompounds of Formula I, wherein the furanosyl sugar moieties of FormulaI are selected from the group consisting of 3′-deoxyribofuranosyl,2′,3′-dideoxyribofuranosyl, arabinofuranosyl, 3′-deoxyarabinofuranosyl,xylofuranosyl, 2′-deoxyxylofuranosyl, and lyxofuranosyl.

The following specific compounds are also novel:P¹-(6-mercaptopurineriboside 5′-)P⁴-(uridine 5′-)tetraphosphate,P¹-(6-mercaptopurineriboside 5′-)P⁴-(2′-deoxyuridine 5′-)tetraphosphate,P¹-(4-thiouridine 5′-)P⁴-(arabinocytidine 5′-)tetraphosphate,P¹-(2′-deoxyadenosine 5′-)P⁴-(6-thiohexylpurineriboside or5′-)tetraphosphate, P¹-(6-octyloxypurineriboside 5′-)P⁴-(uridine5′-)tetraphosphate, P¹-(arabinoadenosine-5′)P⁴-(uridine5′-)tetraphosphate, P¹-(lyxofuranosylthymine 5′-)P⁴-(uridine5′-)tetraphosphate, and P¹-(xylofuranosyluracil5′-)P⁴-(uridine-5′-)tetraphosphate.

Compounds encompassed by the present invention can be prepared bycondensation of a nucleoside mono-, di-, or triphosphate, activated witha condensing agent such as, but not limited to, carbonyldiimidazole ordicyclohexylcarbodiimide, with a second molecule of the same or adifferent mono-, di-, or triphosphate to form the desired dinucleotidepolyphosphate. Another method of preparation is the sequentialcondensation of a nucleoside phosphate, activated as above, with anon-nucleoside mono-, di- or polyphosphate moiety, such as, but notlimited to, a monophosphate or pyrophosphate anion to yield the desireddinucleotide polyphosphate, the non-isolated intermediate in such a casebeing a mononucleotide polyphosphate. Yet another preparative approachis the sequential condensation of a mono-, di- or polyphosphate moiety,activated as mentioned above, or in the form of an acid halide or otherderivative reactive toward nucleophilic displacement, with a nucleosidephosphate or polyphosphate to yield the desired dinucleotidepolyphosphate. The desired dinucleotide polyphosphate can be formed bymodification of a pre-formed dinucleotide polyphosphate by substitutionor derivatization of a moiety or moieties on the purine, pyrimidine orcarbohydrate ring. Nucleoside phosphates used as starting materials arecommercially available, or can be made from the correspondingnucleosides by methods well known to those skilled in the art. Likewise,where nucleosides are not commercially available, they can be made bymodification of other readily available nucleosides, or by synthesisfrom heterocyclic and carbohydrate precursors by methods well known tothose skilled in the art (WO 96/40059, WO 96/02554A1, WO-A-9815563, andWO 98/55494; Theoclitou, et al., J. Chem. Sot. Perkin Trans. I,2009-2019 (1996); Guranowski, et al., Nucleosides and Nucleotides14,731-734 (1995); Visscher, et al., Nucleic Acids Research 20,5749-5752 (1992); Holler, et al., Biochemistry 22, 4924-10 4933 (1983);Orr, et al., Biochem. Pharmacol. 673-677 (1988); Plateau, et al.,Biochemistry 24, 914-922 (1985); Hagmeier, et al., J. Chromatography237, 174-177 (1982); Scheffzek, et al., Biochemistry 35, 9716-9727(1996); Stridh, et al., Antiviral Res., 97-105 (19861); Tarasova, etal., Chem. Abs. 110, 154770 (1988); Hata, et al., Chem Lett., 987-990(1976); Huhn, et al., 28, 1959-1970 (1993); Tumanov, et al., Chem. Abs.109-6867d (1987); Pintor, et al., Molecular Pharmacology 51, 277-284(1997); and U.S. Pat. Nos. 4,855,304; 5,635,160; 5,495,550; and5,681,823).

Those having skill in the art will recognize that the starting materialscan be varied and additional steps employed to produce compoundsencompassed by the present invention. In some cases, protection ofcertain reactive functionalities is useful to achieve some of the abovetransformations. In general, the need for such protecting groups as wellas the conditions necessary to attach and remove such groups will beapparent to those skilled in the art of organic synthesis.

The compounds of the present invention also encompass their non-toxicpharmaceutically acceptable salts, such as, but not limited to, analkali metal salt such as lithium, sodium or potassium; an alkalineearth metal salt such as magnesium or calcium; or an ammonium orquaternary ammonium salt, e.g., NX₄ ⁺ (wherein X is C₁₋₄).Pharmaceutically acceptable salts are salts that retain the desiredbiological activity of the parent compound and do not impart undesiredtoxicological effects. The present invention also encompasses theacylated prodrugs of the compounds disclosed herein. Those skilled inthe art will recognize various synthetic methodologies, which can beemployed to prepare non-toxic pharmaceutically acceptable salts andacylated prodrugs of the disclosed compounds.

Though the compounds of the present invention are primarily concernedwith the treatment of human subjects, they can also be employed for thetreatment of other mammalian subjects such as dogs and cats forveterinary purposes.

The pharmaceutical utility of compounds of this invention is indicatedby the inositol phosphate assay for P2Y₂ and other P2Y receptoractivity. This widely used assay, as described in Lazarowski, etal.(Brit. J. Pharm. 116, 1619-27 (1995)), relies on the measurement ofinositol phosphate formation as a measurement of activity of compoundsactivating receptors linked via G-proteins to phospholipase C.

The efficacy of these compounds is reflected in their ability tofacilitate removal of pathological fluid accumulation in the sub-retinaland intra-retinal spaces, associated with edematous retinal disordersincluding retinal detachment and retinal edema. The effective dose willdepend on characteristics of the individual patient, activity of thespecific compound employed, mode of administration, and characteristicsof the disease or disorder, and can be determined by those skilled inthe art.

Dosage levels to remove extraneous fluid within intra-retinal orsubretinal spaces are of the range of 10 μg/eye to 10 mg/eye, preferablyin the range 50 μg/eye to 6 mg/eye, and most preferably 0.1 mg/eye to 4mg/eye.

Administration of Compounds

The active compounds disclosed herein can be administered to the eyes ofa patient by any suitable means, but are preferably administered byadministering a liquid or gel suspension of the active compound.Alternatively, the active compounds can be applied to the eye vialiposomes. Further, the active compounds can be infused into the tearfilm via a pump-catheter system. Another embodiment of the presentinvention involves the active compound contained within a continuous orselective-release device, for example, membranes such as, but notlimited to, those employed in the Ocuser™ System (Alza Corp., Palo Alto,Calif.). As an additional embodiment, the active compounds can becontained within, carried by, or attached to contact lenses that areplaced on the eye. Another embodiment of the present invention involvesthe active compound contained within a swab or sponge that can beapplied to the ocular surface. Another embodiment of the presentinvention involves the active compound contained within a liquid spraythat can be applied to the ocular surface. Another embodiment of thepresent invention involves an injection of the active compound directlyinto the lachrymal tissues or onto the eye surface.

The active compounds disclosed herein can be administered byintravitreal, systemic, or topical administration. Intravitrealadministration is a preferred route of administration. Intravitrealadministration comprising: single or multiple intravitreal injections;administration directly into the vitreal chamber during surgeryseparately or in conjunction with intraocular irrigation solutions, orother similar solutions or devices routinely used during vitreoretinalsurgery; administration via liposomes or other suitable pharmaceuticalcarriers; administration via continuous or selective-releaseintravitreal-implantable devices, including, but not limited to,Ocusert™ (Alza Corp., Palo, Alto, Calif.) and Vitrasert (Bausch andLomb, Inc., Rochester, N.Y.). The vitreous concentration of the activecompound ranges from 1-500 micromolar, and preferably, 2-200 micromolar.

In one embodiment of the invention, the active compound is injected inthe form of an aqueous pharmaceutical composition into the vitreous in atotal amount between about 0.10 milligrams and about 4.0 milligrams pereye. The aqueous pharmaceutical composition contains an active compoundor a pharmaceutically acceptable salt thereof such as a sodium,potassium, lithium or tetraalkyl ammonium salt. The aqueouspharmaceutical composition in general has an osmolarity between about250 and 350 mOsm, and pH between about 5.0 and 9.0; and preferably hasan osmolarity between about 280 and 300 mOsm, and pH between about 7.0and 7.6. The intravitreal injection can be performed by single ormultiple intravitreal injections at injection volumes of 1-200 μl;preferably 5-100 μl. When the injection volume is small such as 1-25 μl,the pharmaceutical composition can have a broader osmolarity and pHrange. When the injection volume is large such as 100-200 μl, thepharmaceutical composition preferably has a narrower osmolarity and pHrange such as about 280-300 mOsm, and about 7.0-7.6 pH.

The intravitreal solution containing the active compound optionallycontains a physiologically compatible vehicle, as those skilled in theophthalmic art can select using conventional criteria. The vehicles areselected from the known ophthalmic vehicles which include, but are notlimited to, saline solution, water soluble polyethers such aspolyethylene glycol, polyvinyls such as polyvinyl alcohol and povidone,cellulose derivatives such as methylcellulose and hydroxypropylmethylcellulose, petroleum derivatives such as mineral oil and whitepetrolatum, animal fats such as lanolin, polymers of acrylic acid suchas carboxypolymethylene gel, vegetable fats such as peanut oil andpolysaccharides such as dextrans, and glycosaminoglycans such as sodiumhyaluronate and salts such as sodium chloride and potassium chloride.The preferred embodiment is an intravitreal solution comprising activecompound and saline at neutral pH and physiological osmolarity.

The topical solution containing the active compound can also contain aphysiologically compatible vehicle, as those skilled in the ophthalmicart can select using conventional criteria. The vehicles are selectedfrom the known ophthalmic vehicles which include, but are not limitedto, saline solution, water soluble polyethers such as polyethyleneglycol, polyvinyls such as polyvinyl alcohol and povidone, cellulosederivatives such as methylcellulose and hydroxypropyl methylcellulose,petroleum derivatives such as mineral oil and white petrolatum, animalfats such as lanolin, polymers of acrylic acid such ascarboxypolymethylene gel, vegetable fats such as peanut oil andpolysaccharides such as dextrans, and glycosaminoglycans such as sodiumhyaluronate and salts such as sodium chloride and potassium chloride.

In addition to the topical method of administration described above,there are various methods of administering the active compounds of thepresent invention systemically. One such means would involve an aerosolsuspension of respirable particles comprised of the active compound,which the subject inhales. The active compound would be absorbed intothe bloodstream via the lungs or contact the ocular tissues via thenasolacrimal ducts, and subsequently contact the retinal pigmentepithelial cells in a pharmaceutically effective amount. The respirableparticles can be liquid or solid, with a particle size sufficientlysmall to pass through the mouth and larynx upon inhalation; in general,particles ranging from about 1 to 10 microns, but more preferably 1-5microns, in size are considered respirable.

Another means of systemically administering the active compounds to theeyes of the subject would involve administering a liquid/liquidsuspension in the form of eye drops or eye wash or nasal drops of aliquid formulation, or a nasal spray of respirable particles that thesubject inhales. Liquid pharmaceutical compositions of the activecompound for producing a nasal spray or nasal or eye drops can beprepared by combining the active compound with a suitable vehicle, suchas sterile pyrogen free water or sterile saline by techniques known tothose skilled in the art.

Other means of systemic administration of the active compound wouldinvolve oral administration, in which pharmaceutical compositionscontaining compounds of Formula I are in the form of tablets, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsion,hard or soft capsules, or syrups or elixirs. Compositions intended fororal use can be prepared according to any method known to the art forthe manufacture of pharmaceutical compositions and such compositions cancontain one or more agents selected from the group consisting of:sweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnontoxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients include, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for example,starch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate can be employed. Formulations fororal use can also be presented as hard gelatin capsules wherein theactive ingredient is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, peanut oil, liquid paraffin or olive oil.

Additional means of systemic administration of the active compound tothe eyes of the subject would involve a suppository form of the activecompound, such that a therapeutically effective amount of the compoundreaches the eyes via systemic absorption and circulation.

Further means of systemic administration of the active compound wouldinvolve direct intra-operative instillation of a gel, cream, or liquidsuspension form of a therapeutically effective amount of the activecompound.

The method of the present invention is useful to enhance the effects ofsurgery, pharmacotherapy, and adjunctive agents used to treat and managedisorders associated with retinal detachment and retinal edema. Surgicalapproaches include scleral buckle, pneumatic retinopexy, maculartranslocation and vitrectomy. Pharmacotherapeutic agents such ascorticosteroids and acetazolamide have been used to manage macularedema.

High doses may be required for some therapeutic agents to achieve levelsto effectuate the target response, but may often be associated with agreater frequency of dose-related adverse effects. Thus, combined use ofthe compounds of the present invention with agents commonly used totreat retinal detachment and retinal edema permits relatively lowerdoses of such agents resulting in a lower frequency of adverse sideeffects associated with long-term administration of such therapeuticagents. Thus, another indication of the compounds in this invention isto reduce adverse side effects of drugs used to treat retinal detachmentand retinal edema, such as the development of systemic effects withacetazolamide.

The present invention also provides a pharmaceutical formulationcomprising a P2Y agonist such as P¹-(2′-deoxycytidine 5′-)P⁴-(uridine5′-)tetraphosphate, or a pharmaceutically acceptable salt thereof, in apharmaceutically acceptable carrier, in the form of an aqueous, a gel, agel-like, or a solid formulation. Pharmaceutically acceptable salts aresalts that retain the desired biological activity of the active compoundand do not impart undesired toxicological effects. Pharmaceuticallyacceptable salts include alkali metal salts such as lithium, sodium orpotassium salts, alkaline earth metal salts such as magnesium or calciumsalts; or ammonium or tetraalkyl ammonium salts, i.e., NX₄ ⁺ (wherein Xis C₁₋₄). The pharmaceutically acceptable carrier is a physiologicallycompatible vehicle, which includes, but is not limited to, aqueouselectrolyte solutions, polyethers, polyvinyls, polymers of acrylic acid,lanolin, and glucosaminoglycans.

When the pharmaceutical formulation is in the form of an aqueoussolution, it generally comprises physiologically safe excipientsformulated to osmolarity between 250-350 mOsm and pH 5-9; preferably280-300 mOsM and pH 7.0-7.6. When the pharmaceutical formulation is inthe form of a gel or gel-like formulation, it is preferably a hyaluronicacid or hyaluronic acid-containing formulation approved for intraocularsurgical use. When the pharmaceutical formulation is in the form of asolid formulation, it is preferably a lyophilized powder, liposome or abiodegradable polymer.

The pharmaceutical formulation optionally comprises an intraocularirrigation solution approved for surgical use.

The present invention also provides novel compositions ofP¹-(2′-deoxycytidine 5′-) P⁴-(uridine 5′-)tetraphosphate, tetrasodium,tetralithium, tetrapotassium, tetrammonium and tetra(quarternaryammonium) salts. Examples of quaternary ammonium salts includetetraalkylammonium salts, trialkylphenylammonium salts,dialkyldiarylammonium salts, tribenzylalkylammonium salts, etc. Thesetetra-salt compositions are advantageous in that they are readilypurified by aqueous ion chromatography in which only volatile organicsolvents and water are used. They have a high degree (>90%) of purity,thus are suitable for pharmaceutical use. These tetraammonium and tetramonovalent alkali metal salts are more resistant to hydrolysis than themono-, di-, or tri-salts, therefore, they provide an improved stabilityand a longer shelf life for storage. In addition, these salts are easilyhandled as fluffy, white solids, compared to an oil or gum as with someamine salts.

The tetrasodium, tetralithium, and tetrapotassium salts ofP¹-(2′-deoxycytidine 5′-) P⁴-(uridine 5′-)tetraphosphate arenon-irritating to the lung and eyes. Other cations may be irritating tothe lungs, eyes, and other mucosal epithelia, or are otherwise not welltolerated by the human body. The tetrasodium, tetralithium, andtetrapotassium salts are preferred.

The invention is illustrated further by the following examples, whichare not to be construed as limiting the invention in scope or spirit tothe specific procedures described in them.

EXAMPLES Example 1

Localization of P2Y₂-Receptor mRNA in Retina and RPE

Cellular localization of P2Y₂-receptor mRNA in fresh frozencross-sections of albino rabbit retina/RPE/choroid tissue wasinvestigated by using nonisotopic in situ hybridization techniques. FIG.1 shows a representative in situ hybridization result from antisense andsense digoxigenin (DIG)-labeled riboprobes engineered based on the P2Y₂receptor mRNA sequence. Hybridization of antisense and sense riboprobeswas visualized by immunohistochemistry using alkalinephosphatase-conjugated anti DIG antibody, and DIG-specific signal wasdetected using a chromophore reaction against the alkaline phosphatase,yielding purple/black staining. The tissues were also counterstainedwith nuclear fast red. The control sense probe (right) shows no specificlabeling. Labeling with the anti-sense probe showed P2Y₂ receptor mRNAlocalization in scattered nuclei in the ganglion cell and inner nuclearlayers and through the inner segment layer of photoreceptors. Stronglabeling throughout the RPE was also detected, and in endothelial cellsof the choroidal blood vessels.

Example 2

Effects of Synthetic P2Y₂ Agonist UP₄dC on Cloned Human P2Y₂ Receptors

The dinucleotide, [P¹-(uridine 5-)-P⁴-(2′-deoxycytidine5′-)tetraphosphate tetrasodium salt](UP₄dC) INS37217, was tested for itsactivity (potency, efficacy, and selectivity) at cloned human P2Yreceptor subtypes, which were stably expressed in 1321N1 astrocytomacells. Activity was assessed using two in vitro indices of cellactivation: 1) mobilization of intracellular calcium stores, and 2)accumulation of [³H]-inositol phosphates ([3H]-IP). UP₄dC was evaluatedfor activity in both assays against cells expressing the P2Y₁, P2Y₂,P2Y₄, or P2Y₆ receptors.

UTP and UP₄dC induced mobilization of cytosolic calcium in 1321N 1astrocytoma cells expressing human P2Y₂ (FIG. 2) receptors with EC₅₀values of 0.22 μM and 0.8 μM, respectively. The calcium response to 100μM UP₄dC was 100% of the maximal response to UTP at P2Y₂ receptors. Inconclusion, UP₄dC is a full agonist for calcium mobilization at P2Y₂receptors compared to UTP.

UTP and UP₄dC stimulated [3H]-IP accumulation in 1321N1 cells expressinghuman P2Y₂ (FIG. 3) receptors with an EC₅₀ values of 1.1 and 2.2 μM,respectively. The inositol phosphate response to 100 μM UP₄dC wasapproximately that of the maximal response to UTP. In conclusion, UP₄dCis a full agonist for inositol phosphate release at P2Y₂ receptorscompared to UTP in the test system.

Example 3

UP₄dC Stimulates Fluid Absorption in Freshly Isolated RPE Monolayers

Fluid transport across freshly isolated, intact bovine and human fetalRPE monolayers was studied using a modified capacitance probe technique(Frambach, et al., Biophys. J. 47(4):547-52 (1985); Hughes, et al., JGen. Physiol. 83(6):875-99 (1984)).

The RPE was mounted vertically in a modified Ussing chamber such thatapical and basolateral membranes were separately exposed to Ringer'ssolutions held in bathing reservoirs. Stainless steel capacitive probeswere lowered into the apical and basolateral bathing reservoirs to sensethe capacitance of the air gap between the probe and fluid meniscus.Fluid transport rate J_(v) (μL cm⁻² hr⁻¹) was determined by monitoringfluid movement-induced changes in the air gap capacitance at the apicaland basolateral baths.

Representative effects of agonist on J, in human fetal RPE are shown inFIG. 4. Positive J_(v) values reflect fluid absorption(apical-to-basolateral) and negative J_(v) values reflect fluidsecretion (basolateral-to-apical). In the experiment shown in FIG. 4,control fluid movement across the freshly isolated human fetal RPEmonolayer is absorptive at a rate of ˜5 μL cm⁻² hr⁻¹. The addition of 50μM agonist to Ringer's solution bathing the apical membrane elicited atransient increase in fluid absorption to ˜40 μL cm⁻² hr⁻¹ beforereturning back to pre-stimulated levels. During the 1-hour treatmentperiod, UP₄dC increased total fluid absorption by approximately a factorof three.

Although the RPE is normally a fluid-absorbing epithelium, fluidsecretion has occasionally been observed in freshly isolated RPEpreparations. It has been postulated that fluid secretion in vivo may bea normal component of RPE physiology under certain conditions, such asfollowing a transition between dark and light, or under pathologicalconditions, such as in serous retinal detachments. FIG. 5 shows that ina freshly isolated bovine RPE monolayer in which J_(v) secretion isobserved under control conditions, the agonist can reverse the directionof fluid transport to absorption. The effects of agonist are reversibleupon returning to control Ringer's solution. Such an effect of agonistin vivo will offer therapeutic potential in the treatment of serousretinal detachments, such as central serous retinopathy, in whichabnormal RPE-mediated fluid secretion is postulated to mediate theeffects of transport of choroidal fluid into the subretinal space.

Example 4

Comparisons of Metabolic Stability of UP₄dC and UTP in Freshly IsolatedRetinal Tissue

The metabolism rate of UP₄dC and UTP in freshly isolated pig retinaltissue was determined using a high performance liquid chromatography(HPLC) method with UV-coupled detection. Freshly isolated retinaltissues of uniform size were isolated from euthanized young pigs (2-3months) and retinal tissues were individually placed in an incubationchamber at 37° C. After an equilibration period of 30 minutes, eachtissue was spiked with 100 μM UP₄dC or UTP in physiological buffer, andincubated for 0.5, 1, 2, and 4 hours. An aliquot of buffer from eachchamber well was then processed for UV-coupled HPLC detection forchromatograms of the parent UP₄dC and UTP compounds at each time pointto track the metabolism rates of each parent compound. FIG. 6 shows thatUP₄dC has a four-fold greater metabolic half-life than UTP under theseexperimental conditions.

Example 5

Effects of Subretinal and Intravitreal UP₄dC in Rabbit Models Methods

Surgical Procedure for Inducing Subretinal Blebs

New Zealand White rabbits weighing approximately 1.5 kg (2-3 month old)were anesthetized with an intramuscular injection of 0.3 ml ketaminehydrochloride (100 mg/ml) and 0.5 ml xylazine hydrochloride (100 mg/ml)per kilogram body weight. Ketamine hydrochloride was added as needed.For experiments requiring observation of the fundus, the pupil wasdilated with scopolamine hydrobromide 0.25%, cyclogyl 1% andphenylephrine hydrochloride 2.5% eyedrops.

One local retinal detachment was created in each eye. A wire lidspeculum was placed and a segmental conjunctival peritomy (ofapproximately 2 clock hours) was made at the 3 and 9 o'clock positions.Two scleral incisions were made with a 19 gauge MVR-blade 0.5 mmposterior to the limbus through the ciliary body. A self-retainingplanoconcave contact lens was placed on the corneal surface. Achandelier light, which was used for illumination, (Grieshaber & Co., AGSchaffhausen, Switzerland) was carefully guided through one of thesclerotomy sites into the vitreous cavity to avoid touching the lens.

Retinal detachments were made with a beveled 36 gauge retinal needle(Grieshaber & Co., AG Schaffhausen, Switzerland) attached by anextension tube to a 1 ml syringe that was driven by a calibrated,mechanical syringe pump (model 351, Sage Instruments, Cambridge, Mass).Under direct observation with an operating microscope, the retinalneedle was inserted through the second sclerotomy and slowly advanced toeither the nasal or temporal myelin wing. These sites were selected forinjection as the myelin wing gives additional structural support whencompared to the adjacent areas of thin, avascular retina. Theintraocular pressure was maintained at a low level to allow a slowhydrodissection of the fragile retina from the RPE. The tip of the 36gauge needle was carefully inserted under the myelin wing. A localizeddome-shaped detachment of the retina was created by using a mechanicalsyringe pump to inject ˜50 μl of phosphate buffered saline fluid intothe subretinal space. The instruments were removed from the eye and thesclerotomy sites remained open to keep the intraocular pressureconstant. Although these experimental retinal detachments have a verysmall retinal hole, they behave functionally like non-rhegmatogenousretinal detachments and have been used to study mechanisms of subretinalfluid reabsorption.

Injection Solution

Modified phosphate buffered saline (MPBS) solution, used for allsubretinal and intravitreal injections, was composed of 13.6 mM Na₂HPO₄,6.2 mM NaH₂PO₄, 130.5 mM NaCl and 5 mM KCl, had an osmolarity of ˜300mOsm and a pH 7.2. UP₄dC (MW 862) was added to the MPBS solution toachieve a target drug concentration of 12 mM, 1.4 mM, 1.0 mM or 0.15 mM.The experimental and control solutions were kept at equal osmolarity.For concentrations of 1 mM UP₄dC or less, an appropriate amount of NaClwas added to the MPBS solution to compensate for the osmolaritycontribution of UP₄dC (1 mM UP₄dC contributes ˜4-5 mOsm). Forconcentrations greater than 1 mM UP₄dC, solution isotonicity wasmaintained by reducing an equal osmolar of NaCl in the MPBS solution inplace of the addition of UP₄dC. All experimental and control solutionsfor each dosing cohort were formulated such that the final osmolaritieswere ±2 mOsm of each other. Sterile solutions were provided andevaluated under investigator-masked conditions.

Study Design

For each animal, one eye served as the experimental eye and thecontralateral eye served as control. UP₄dC was delivered eithersubretinally or intravitreally to evaluate its effects on reabsorptionof subretinal fluid in experimentally induced retinal detachments. Inthe first series of experiments, MPBS solution with or without UP₄dC (1mM) was injected into the subretinal space. In the second series ofexperiments, a subretinal bleb containing MPBS solution was created, andthen 50 μl of MPBS solution with or without UP₄dC (12 mM, 1.4 mM, and0.15 mM) was administered into the vitreous cavity with a 100 μlHamilton syringe adjacent to the subretinal bleb. The surgeon was maskedwith respect to the content of the administered solutions.

Postoperative Observation

The corneal epithelium was protected with a layer of methylcellulose tomaintain corneal clarity. Fundus photographs were obtained with a funduscamera (TRC-W, TOPCON, Japan) in selected cases. The observer determinedby indirect ophthalmoscopy the initial bleb size, and bleb size at30-minute intervals for 3 hours. The vertical and horizontal dimensionsof each subretinal bleb were recorded in disc diameters, using theadjacent optic disc as a reference marker. (The mean reference diameterof the optic disc is approximately 1 mm, as previously determined undera microscope in 10 enucleated eyes from albino rabbits). Bleb size ateach evaluation time point was first quantified by multiplying thevertical and horizontal dimensions, and then expressing the resultantvalue as a dimensionless ratio relative to the initial size of eachbleb. This dimensionless (normalized) bleb size at each evaluation timepoint was then plotted and analyzed as a function of time.

Results

Effects of Subretinal and Intravitreal UP₄dC

In the first series of experiments, an isotonic MPBS solution containing1 mM UP₄dC was injected directly into the subretinal space. Thecontralateral eye received a subretinal injection of isotonic MPBSsolution alone. FIG. 7 shows that UP₄dC-containing subretinal blebsresolved significantly faster than blebs containing MPBS solution alone.Reattachment of the retina was observed at 120-150 minutes in theUP₄dC-treated blebs, whereas the control subretinal blebs did notresolve over the 3 hour observation period. There was a significantdifference in subretinal reabsorption at 30, 60, and 90 minutes usingrepeated measures ANOVA (p<0.05).

In the second series of experiments, subretinal blebs were first inducedwith MPBS solution and then followed immediately with a 50 μlintravitreal injection of MPBS solution with or without UP₄dC (12, 1.4,and 0.15 mM)just adjacent to the bleb. FIGS. 8A & B shows that the twohigher doses of UP₄dC significantly enhanced the rate of subretinal blebreabsorption and retinal reattachment when compared with vehicle. Therewas a statistically significant difference in the reabsorption ratebetween the eye treated with UP₄dC and the vehicle alone at 30, 60, and90 minutes (p<0.05). UP₄dC-treated eyes showed near complete retinalreattachment by ˜90 min, whereas control blebs had not completelyreabsorbed at 180 min.

FIG. 8C shows the reabsorption rate of subretinal bleb in eyes treatedwith 0.15 mM intravitreal UP₄dC or vehicle alone. There was nostatistically significant difference in the reabsorption of subretinalfluid at any time point in eyes with 0.15 mM UP₄dC compared to vehiclealone.

Example 6

Effects of UP₄dC on Retinal Reattachment in Rat Models Methods

In Vivo Preparation: Rat Study Design

Retinal detachments were created in Long-Evans female rats by injecting2-3 μl of modified phosphate buffer saline (MPBS) Ringer solution intothe subretinal space; only one eye per rat was used. Using a CCD camera,images of the subretinal blebs were obtained at one min intervals forseveral hours. The acquisition of images is described in further detailbelow. In the control part of each experiment (at 0 to 30 min followingcreation of the retinal detachment), apparent bleb size reached asteady-state size, which remained unchanged during the course ofanesthesia (several hours). MPBS solutions with or without UP₄dC (5 mM)were formulated and injected (3 μl) into the vitreous of the rat eyeunder masked and randomized conditions. The vials and their contentswere indistinguishable. After vitreous injection, the apparent bleb sizeeither increased or decreased monotonically or was constant over thenext 60 min as judged by the experimenter using the seven rank scale(0±3) illustrated in FIG. 9. Ranks were assigned by observing the changein apparent bleb size between 30-90 minutes after drug or placebovitreous injection. Animals were re-anesthetized the next day and aseparate estimate of rank was obtained. A rank of negative 3 means thatthe retinal bleb was apparently flattened. A rank of positive threemeans that the bleb approximately doubled in size. A zero rank meansthat the apparent bleb size was unchanged over time. After all of theexperiments were completed, the content of each vial was unmasked andcompared with the experimenter's conclusions based on the observation ofimages obtained between 30 and 90 minutes and at one day followingadministration of drug or placebo.

Results

The effects of intravitreal UP₄dC on retinal reattachment in 12 rats (1eye/animal was dosed) are shown in FIG. 9. The experiments were carriedout in a masked fashion to provide a rigorous and objective evaluationof the effects of UP₄dC on fluid reabsorption from experimentallyproduced subretinal blebs. In these experiments, after the creation ofblebs, drug or placebo solutions were injected into the vitreous of therat eye in a masked fashion (vials and their contents wereindistinguishable). After all 12 eyes were scored, the key was unmaskedand compared with the summarized results based on the observations at 1and 24 hours. The results summarized in FIG. 9 show a significantdifference (p<0.05) between UP₄dC (open bars) and vehicle control(placebo, solid bars) on the scoring of subretinal blebs. After one hourof treatment, the UP₄dC-treated eyes all showed a decrease in bleb size,whereas the control eyes all showed an increase in bleb size. The nextday, the subretinal blebs from the UP₄dC-treated eyes had almostcompletely disappeared, whereas the subretinal blebs from thevehicle-treated eyes remained essentially unchanged. In 4 out of 6UP₄dC-treated eyes, the retina appeared completely flat at the 24-hrtime point.

Example 7

Primary Treatment for Subject with Macula-Off Rhegmatogenous RetinalDetachment

A patient presents with sudden onset of loss of central vision and isdiagnosed with macula-off rhegmatogenous retinal detachment with asingle break in the superior retina that is less than one clock hour insize. The patient's conjunctiva (cul de sacs) is sterilized with topicalBetadine and by scrubbing and draping the face and lashes and lids.Local anesthesia is given via subconjunctival injection of xylocaine.

A patient is then given a single, slowly administered 50 μL-intravitrealinjection of a sterile pharmaceutical composition by insertion of a 29or 30 gauge needle from a 0.25 cc or 0.50 cc tuberculin syringe throughthe sclera in the pars plana region of the eye. The pharmaceuticalcomposition consists of a metabolically resistant P2Y₂ receptor agonistformulated to isotonicity (280-300 mOsm) and physiological pH (7.0-7.5)in saline. The amount of active compound (P2Y agonist) administered tothe eye is in the range of about 0.10 milligrams and about 4.0milligrams.

The patient's eyes are bilaterally patched and the patient remainsrested in a horizontal position for 4 hours, at which time the eyes areexamined for retinal reattachment. If the retina has not completelyreattached at the four hour time point, the patient's eyes remainbilaterally patched until the next day (20-24 hours post dosing), atwhich point the retina is reexamined for reattachment. Following retinalreattachment, the retinal tear is suitably treated by conventionalmethods such as cryotherapy or laser photocoagulation.

Example 8

The Structure Elucidation of P¹-(2′-Deoxycytidine 5′-)P⁴-(uridine 5′-)Tetraphosphate Tetrasodium Salt

Due to the lack of adequate spectroscopic data of nonadenylateddinucleotides in the literature, a full structure elucidation ofP¹-(2′-deoxycytidine 5′-)P⁴-(uridine 5′-) tetraphosphate tetrasodiumsalt was performed by employing modern analytical techniques. Themolecular mass was determined by mass spectrometry to be 861 [m/z 862,(M+H⁺)], confirming the molecular formula C₁₈H₂₃N₅O₂₁P₄.4Na. Karl Fishermoisture analysis gave a value of 6.598% H₂O and further confirmation ofthe molecular formula was obtained from elemental analysis: calculatedfor Na=9.97, found=9.99%, based on the molecular formula:C₁₈H₂₃N₅O₂₁P₄.4Na.3.4H₂O (FW=922.1 g/mol). The infrared spectrum showeda broad signal at 3417 cm⁻¹ and signals at 1677 and 1651 cm⁻¹,indicating the presence of hydroxyl (O—H stretch) and carbonyl (C═Ostretch) functional groups. In addition, a phosphate (P═O stretch) wasobserved at 1249 cm⁻¹. The UV spectrum in water displayed a λ_(max) of265.8 nm.

The NMR spectra are: ¹H NMR (D₂O, TMS) δ 2.16 (m, 1H), 2.30 (m, 1H),4.08 (m, 1H), 4.09 (m, 1H), 4.13 (m, 1H), 4.14 (m, 2H), 4.16 (m, 1H),4.27 (m, 2H), 4.49 (m, 1H), 5.85 (d, J=8.2 Hz, 1H), 5.86 (d, J=4.8 Hz,1H), 6.01 (d, J=7.5 Hz, 1H), 6.19 (m, 1H), 7.79 (d, J=8.2 Hz, 1H), 7.81(d, J=7.5 Hz, 1H); ¹³C NMR (D₂O, TMS) δ 39.3, 64.9 (d, J=4.5 Hz), 65.2(d, J=4.7 Hz), 69.4, 70.5, 73.6, 83.2 (d, J=9.4 Hz), 85.3 (d, J=10 Hz),85.7, 88.0, 96.4, 102.5, 141.4, 141.4, 151.6, 157.4, 165.9, 166.0; ³¹PNMR (D₂O, H₃PO₄) δ−21.5 (m), −10.3 (m).

The invention, and the manner and process of making and using it, arenow described in such full, clear, concise and exact terms as to enableany person skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the scope of the present invention as setforth in the claims. To particularly point out and distinctly claim thesubject matter regarded as invention, the following claims conclude thisspecification.

What is claimed is:
 1. A pharmaceutical formulation comprisingP¹-(2′-deoxycytidine 5′-)P⁴-(uridine 5′-) tetraphosphate, or apharmaceutically acceptable salt thereof, in a pharmaceuticallyacceptable carrier, in the form of an aqueous, a gel, a gel-like, or asolid formulation.
 2. The pharmaceutical formulation according to claim1, wherein the pharmaceutical formulation is in the form of an aqueoussolution and comprises physiologically safe excipients formulated toosmolarity between 250-350 mOsm and pH 5-9.
 3. The pharmaceuticalformulation according to claim 2, wherein the pharmaceutical formulationis sterile and contains P¹-(2′-deoxycytidine 5′-)P⁴-(uridine 5′-)tetraphosphate, or a pharmaceutically acceptable salt thereof, in theamount of 0.1-0.5% by weight.
 4. The pharmaceutical formulationaccording to claim 1, wherein said pharmaceutical formulation furthercomprises an intraocular irrigation solution approved for surgical use.5. The pharmaceutical formulation according to claim 1, wherein said gelor gel-like formulation is selected from the group consisting of:hyaluronic acid and hyaluronic acid-containing formulations approved forintraocular surgical use.
 6. The pharmaceutical formulation according toclaim 1, wherein said solid formulation is selected from the groupconsisting of: lyophilized powder, liposome and biodegradable polymer.7. The pharmaceutical formulation according to claim 1, wherein saidpharmaceutically acceptable carrier is a physiologically compatiblevehicle selected from the group consisting of: aqueous electrolytesolutions, polyethers, polyvinyls, polymers of acrylic acid, lanolin,and glucosaminoglycans.
 8. A compound selected from the group consistingof P¹-(2′-deoxycytidine 5′-) P⁴-(uridine 5′-)tetraphosphate, tetrasodiumsalt; P¹-(2′-deoxycytidine 5′-)P⁴-(uridine 5′-)tetraphosphate,tetrapotassium salt; P¹-(2′-deoxycytidine 5′-) P⁴-(uridine5′-)tetraphosphate, tetralithium salt; and P¹-(2′-deoxycytidine5′-)P⁴-(uridine 5′-)tetraphosphate, tetraammonium salt.